Overexpression of VIRMA confers vulnerability to breast cancers via the m6A-dependent regulation of unfolded protein response.

Virilizer-like m6A methyltransferase-associated protein (VIRMA) maintains the stability of the m6A writer complex. Although VIRMA is critical for RNA m6A deposition, the impact of aberrant VIRMA expression in human diseases remains unclear. We show that VIRMA is amplified and overexpressed in 15-20% of breast cancers. Of the two known VIRMA isoforms, the nuclear-enriched full-length but not the cytoplasmic-localised N-terminal VIRMA promotes m6A-dependent breast tumourigenesis in vitro and in vivo. Mechanistically, we reveal that VIRMA overexpression upregulates the m6A-modified long non-coding RNA, NEAT1, which contributes to breast cancer cell growth. We also show that VIRMA overexpression enriches m6A on transcripts that regulate the unfolded protein response (UPR) pathway but does not promote their translation to activate the UPR under optimal growth conditions. Under stressful conditions that are often present in tumour microenvironments, VIRMA-overexpressing cells display enhanced UPR and increased susceptibility to death. Our study identifies oncogenic VIRMA overexpression as a vulnerability that may be exploited for cancer therapy.

Macrophage development and activation involve coordinated intron retention in key inflammatory regulators.

Monocytes and macrophages are essential components of the innate immune system. Herein, we report that intron retention (IR) plays an important role in the development and function of these cells. Using Illumina mRNA sequencing, Nanopore direct cDNA sequencing and proteomics analysis, we identify IR events that affect the expression of key genes/proteins involved in macrophage development and function. We demonstrate that decreased IR in nuclear-detained mRNA is coupled with increased expression of genes encoding regulators of macrophage transcription, phagocytosis and inflammatory signalling, including ID2, IRF7, ENG and LAT. We further show that this dynamic IR program persists during the polarisation of resting macrophages into activated macrophages. In the presence of proinflammatory stimuli, intron-retaining CXCL2 and NFKBIZ transcripts are rapidly spliced, enabling timely expression of these key inflammatory regulators by macrophages. Our study provides novel insights into the molecular factors controlling vital regulators of the innate immune response.

Drosophila Syncrip modulates the expression of mRNAs encoding key synaptic proteins required for morphology at the neuromuscular junction.

Localized mRNA translation is thought to play a key role in synaptic plasticity, but the identity of the transcripts and the molecular mechanism underlying their function are still poorly understood. Here, we show that Syncrip, a regulator of localized translation in the Drosophila oocyte and a component of mammalian neuronal mRNA granules, is also expressed in the Drosophila larval neuromuscular junction, where it regulates synaptic growth. We use RNA-immunoprecipitation followed by high-throughput sequencing and qRT-PCR to show that Syncrip associates with a number of mRNAs encoding proteins with key synaptic functions, including msp-300, syd-1, neurexin-1, futsch, highwire, discs large, and α-spectrin. The protein levels of MSP-300, Discs large, and a number of others are significantly affected in syncrip null mutants. Furthermore, syncrip mutants show a reduction in MSP-300 protein levels and defects in muscle nuclear distribution characteristic of msp-300 mutants. Our results highlight a number of potential new players in localized translation during synaptic plasticity in the neuromuscular junction. We propose that Syncrip acts as a modulator of synaptic plasticity by regulating the translation of these key mRNAs encoding synaptic scaffolding proteins and other important components involved in synaptic growth and function.

Ultraprecise single-molecule localization microscopy enables in situ distance measurements in intact cells.

Single-molecule localization microscopy (SMLM) has the potential to quantify the diversity in spatial arrangements of molecules in intact cells. However, this requires that the single-molecule emitters are localized with ultrahigh precision irrespective of the sample format and the length of the data acquisition. We advance SMLM to enable direct distance measurements between molecules in intact cells on the scale between 1 and 20 nm. Our actively stabilized microscope combines three-dimensional real-time drift corrections and achieves a stabilization of <1 nm and localization precision of ~1 nm. To demonstrate the biological applicability of the new microscope, we show a 4- to 7-nm difference in spatial separations between signaling T cell receptors and phosphatases (CD45) in active and resting T cells. In summary, by overcoming the major bottlenecks in SMLM imaging, it is possible to generate molecular images with nanometer accuracy and conduct distance measurements on the biological relevant length scales.

Can single molecule localization microscopy detect nanoclusters in T cells?

Nanoclusters of cell surface receptors have been detected with single molecule localization microscopy (SMLM) and are thought to mediate signal transduction. Clustering of the T cell receptor (TCR), for example, was reported to control signalling efficiency and antigen discrimination. However, the ability to detect nanoclusters with SMLM has been questioned. Here, we review the detection limits of SMLM as defined by both the physical limits and data processing, as well as evidence for nanoclusters arising from complementary techniques. We conclude with an outlook of how future data analysis can reveal the implications of molecular self-organization for signalling.

Are stentless valves superior to modern stented valves? A prospective randomized trial.

BACKGROUND: It is presumed that stentless aortic bioprostheses are hemodynamically superior to stented bioprostheses. A prospective randomized controlled trial was undertaken to compare stentless versus modern stented valves. METHODS AND RESULTS: Patients with severe aortic valve stenosis (n=161) undergoing aortic valve replacement (AVR) were randomized intraoperatively to receive either the C-E Perimount stented bioprosthesis (n=81) or the Prima Plus stentless bioprosthesis (n =80). We assessed left ventricular mass (LVM) regression with transthoracic echocardiography (TTE) and magnetic resonance imaging (MRI). Transvalvular gradients were measured postoperatively by Doppler echocardiography to compare hemodynamic performance. There was no difference between groups with regard to age, symptom status, need for concomitant coronary artery bypass surgery, or baseline LVM. LVM regressed in both groups but with no significant difference between groups at 1 year. In a subset of 50 patients, MRI was also used to assess LVM regression, and again there was no significant difference between groups at 1 year. Hemodynamic performance of the 2 valves was similar with no difference in mean and peak systolic transvalvular gradients 1 year after surgery. In patients with reduced ventricular function (left ventricular ejection fraction [LVEF] <60%), there was a significantly greater improvement in LVEF from baseline to 1 year in stentless valve recipients. CONCLUSIONS: Both stented and stentless bioprostheses are associated with excellent clinical and hemodynamic outcomes 1 year after AVR. Comparable hemodynamics and LVM regression can be achieved using a second-generation stented pericardial bioprosthesis. In patients with ventricular impairment, stentless bioprostheses may allow for greater improvement in left ventricular function postoperatively.

Avoidance of hemodilution during selective cerebral perfusion enhances neurobehavioral outcome in a survival porcine model.

INTRODUCTION: The ideal hematocrit (HCT) level during hypothermic selective cerebral perfusion (SCP)--to ensure adequate oxygen delivery without excessive perfusion--has not yet been determined. METHODS: Twenty pigs (26.0+/-2.6 kg) were randomized to low or high HCT management. The cardiopulmonary bypass (CPB) circuit was primed with crystalloid in the low HCT group (21+/-1%), and with donor blood in the high HCT group (30+/-1%). Pigs were cooled to 20 degrees C and SCP was carried out for 90 min. During rewarming, whole blood was added in the low HCT group and crystalloid in the high HCT group to produce equivalent HCT levels by the end of the procedure. Using fluorescent microspheres and sagittal sinus sampling, cerebral blood flow (CBF) and oxygen metabolism (CMRO2) were assessed at baseline, after cooling, at two points during SCP (30 and 90 min), and at 15 min and 2 h post-CPB. In addition, a range of physiological and metabolic parameters, including intracranial pressure (ICP), were recorded throughout the procedure. The animals' behavior was videotaped and assessed blindly for 7 days postoperatively (maximum score=5). RESULTS: HCT levels were equivalent at baseline, 2 h post-CPB, and at sacrifice, but significantly different (p<0.0001) during cooling and SCP. Mean arterial pressure, pH and pCO2, and CMRO2 were equivalent between groups throughout. ICP was similar in the two groups throughout cooling, SCP, and rewarming, but was significantly higher in the low HCT animals after the termination of CPB. CBF was similar at baseline, but thereafter markedly higher in the low HCT group. Neurobehavioral performance was significantly better in the high HCT animals (median score 3.5 vs 4.5 on day 3, and 4.5 vs 4.75 on day 7, p=0.003). CONCLUSIONS: Higher HCT levels for SCP produced a significantly superior functional outcome, suggesting that the higher CBF with a lower HCT may be injurious, possibly because of an increased embolic load.

Laparoscopic repair of a migrated adjustable gastric band connecting tube with colonic erosion.

Laparoscopic adjustable gastric bands are a popular and effective surgical option to treat morbid obesity. The overall complication rate is 10-20% and the most common complication is of 'slippage'. Although other complications such as gastric band migration and erosion have been reported, the phenomenon of a migrated gastric band connecting tube eroding into the colon (after port removal) is seldom reported in the literature. In this article we describe such a case of an incidentally found colonic erosion on colonoscopy and describe the subsequent laparoscopic repair, as well as a review of the literature.

Comparison of survival by allocation to medical therapy, surgery, or heart transplantation for ischemic advanced heart failure.

BACKGROUND: To ascertain survival of ischemic advanced heart failure patients by treatment allocation, we examined the outcome of transplant assessment patients allocated to medical therapy, high-risk conventional surgery, or transplantation. METHODS: Patients were identified from the Papworth transplant database and excluded if primary etiology was not ischemic. Grouping was undertaken according to treatment allocation at initial assessment, and analysis was performed by intention to treat. Survival was computed from the time of assessment and Cox regression used to stratify patients according risk with the Heart Failure Survival Score. RESULTS: From May 1993 to September 2001, a total of 755 patients were admitted for transplant assessment, with 348 (46.1%) identified as having heart failure of ischemic origin. Variables required for calculation of the Heart Failure Survival Score was available in 273 patients (78.4%), and 20 patients (7.3%) were lost to follow-up. Of the remaining 253 patients, 89 (35.2%) were allocated to medical therapy, 32 (12.6%) to surgery, and 132 (52.2%) to transplantation. The relative risk (95% confidence limit) of death compared with medical therapy was 0.62 (0.28, 1.40) for surgery and 0.38 (0.24, 0.61) for transplantation in medium- to high-risk patients. For low-risk patients, the relative risks for death compared with medical therapy were 1.87 (0.63, 5.60) for surgery and 1.97 (0.79, 4.96) for transplantation. CONCLUSIONS: Transplantation improved survival of medium- and high-risk patients compared with medical therapy. In the low-risk group, this was not evident. However, repeated assessment of risk is required because the hazard for death rises steadily after the third year in these patients.

Is routine use of stentless aortic prostheses justified in an elderly (aged > or =75 years) population?

BACKGROUND AND AIM OF THE STUDY: Stentless prostheses in the aortic position produce a superior hemodynamic profile in comparison to that with stented valves. To determine whether routine use of stentless valves in an elderly population is justified, a 10-year retrospective review was performed of a consecutive series of patients aged > or =75 years undergoing stentless aortic valve replacement (AVR). METHODS: Demographic, operative and mortality data were obtained retrospectively. Survivors were interviewed by telephone according to a defined protocol. Univariate and multivariate analysis was used to identify independent predictors of 30-day and overall medium-term mortality. Definitions and analyses were in accordance with joint STS/AATS guidelines. RESULTS: A total of 103 patients (57 males, 46 females; mean age 79.8 years; range: 75-91 years) underwent AVR with a either a Toronto stentless porcine valve (size range: 21-29 mm; n = 74) or an aortic homograft (n = 29). Twenty-eight patients (27%) had either urgent/emergency surgery, 12 (11%) underwent redo surgery, and in 54 cases (52%), the preoperative left ventricular function was significantly impaired (ejection fraction <50%). Forty patients (39%) also underwent concomitant coronary artery bypass grafting. The mean cross-clamp and cardiopulmonary bypass times were 105+/-22 min and 144+/-47 min, respectively. The overall 30-day mortality was 11.6% (n = 12). The 30-day mortality for all elective cases was 5.3%, but for isolated elective AVR was only 2.5%. Using a multivariate model, the only independent predictor of 30-day mortality and medium-term overall mortality was increasing age. The mean follow up period was 3.6 years (range: 0.1-9.3 years), and the Kaplan-Meier actuarial five-year survival was 52%. At follow up, 92% of patients were in NYHA functional classes I and II. CONCLUSION: Stentless AVR in elderly patients is associated with excellent functional and survival outcome in the medium term. Furthermore, in elective cases, age alone should not be a deterrent to the routine use of stentless aortic valves.

Optimal pH strategy for selective cerebral perfusion.

OBJECTIVE: Selective cerebral perfusion (SCP) affords brain protection superior to hypothermic circulatory arrest (HCA) for prolonged aortic arch procedures. Optimal pH strategy for HCA is controversial; for SCP it is unknown. We compared pH strategies during SCP in a survival pig model. METHODS: Twenty juvenile pigs (26+/-2.4 kg), randomized to alpha-stat (n=10) or pH-stat (n=10) management, underwent cooling to 20 degrees C on cardiopulmonary bypass (CPB) followed by 90 min of SCP at 20 degrees C. SCP was conducted with a mean pressure of 50 mmHg and hematocrit of 22.5%. Using fluorescent microspheres and sagittal sinus blood sampling, cerebral blood flow (CBF) and oxygen metabolism (CMRO2) were assessed at the following time points: baseline, after 30 min cooling (20 degrees C), 30 min of SCP, 90 min of SCP, 15 min post-CPB and 2h post-CPB. Visual evoked potentials (VEP) were assessed at baseline and monitored for 2h during recovery. Neurobehavioral recovery (10=normal) was assessed in a blinded fashion for 7 postoperative days. RESULTS: There were no significant differences between the groups at baseline. CBF was significantly higher at the end of cooling, and after 30 and 90 min of SCP in the pH-stat group (P=0.02, 0.007, 0.03). CMRO2 was also higher with pH-stat (P=0.06, 0.04, 0.10). Both groups showed prompt return to values close to baseline after rewarming (P=ns). VEP suggested a trend towards improved recovery in the alpha-stat group at 2h post-CPB, P=0.15. However, there were no significant differences in neurobehavioral score: (alpha-stat versus pH-stat) median values 7 and 7.5 on day 1; 9 and 9 on day 4, and 10 and 10 on day 7. CONCLUSIONS: These data suggest that alpha-stat management for SCP provides more effective metabolic suppression than pH-stat, with lower CBF. Clinically, the better preservation of cerebral autoregulation during alpha-stat perfusion should reduce the risk of embolization.

Composite aortic root replacement in acute type A dissection: time to rethink the indications?

OBJECTIVE: The indications for aortic root replacement in acute type A dissection are unclear. We reviewed the immediate and long-term outcome of consecutive patients in a series in which a low-threshold policy of composite aortic root replacement had evolved. METHODS: From a prospectively compiled aortic surgery database, we identified 162 patients who had either supracoronary interposition grafting, Group A (n=89), or composite root replacement, Group B (n=73) for acute type A dissection. Patients receiving total arch replacements were excluded. Operative and clinical details were analyzed and patient survival was compared to an age and gender matched census cohort. Need for reoperation on the proximal or distal aorta was also noted. Follow-up totaled 795.5 patient-years. RESULTS: Hospital mortality rates were identical in both groups (12.3%: 11 deaths in group A; 9 in group B). Chronic pulmonary disease, diabetes, malperfusion, hemodynamic compromise and aortic root dilatation were independent risk factors for hospital death. Actuarial survival estimates at 1, 5 and 10 years were 79% (71-88%), 64% (53-75%), and 55% (41-68%) for group A, and 79% (70-86%), 73% (62-83%), and 65% (52-78%) for group B (P=0.48). Age and operative patency of the ascending false lumen were independent risk factors for death after hospital discharge. Proximal aortic reoperation was required for four patients in group A and none in group B (P=0.085). CONCLUSION: A strategy of replacement rather than repair of the dissected aortic root for specific indications in type A dissection yielded high survival and low proximal reoperation rates. These results support an aggressive policy of composite root replacement in acute type A dissection.

Translation. An RNA biosensor for imaging the first round of translation from single cells to living animals.

Analysis of single molecules in living cells has provided quantitative insights into the kinetics of fundamental biological processes; however, the dynamics of messenger RNA (mRNA) translation have yet to be addressed. We have developed a fluorescence microscopy technique that reports on the first translation events of individual mRNA molecules. This allowed us to examine the spatiotemporal regulation of translation during normal growth and stress and during Drosophila oocyte development. We have shown that mRNAs are not translated in the nucleus but translate within minutes after export, that sequestration within P-bodies regulates translation, and that oskar mRNA is not translated until it reaches the posterior pole of the oocyte. This methodology provides a framework for studying initiation of protein synthesis on single mRNAs in living cells.

Pyrexia after cardiac surgery: natural history and association with infection.

BACKGROUND: Pyrexia is common after major surgery, and infection is often an important consideration. To investigate the natural history and association with infection, we performed a prospective observational study. METHODS: From November 2000 to January 2001, we studied 219 patients undergoing cardiac surgery screening daily for wound, respiratory, urinary tract, and other infections. Pyrexia was defined as temperature above 37.5 degrees C. RESULTS: Of 219 patients, 7 intraoperative deaths occurred and 1 patient was excluded because of preoperative endocarditis, leaving 211. The mean age (SD) was 64 (10) years, consisting of 172 male patients (81.5%). The proportion pyrexial on days 1, 2, and 5 was 30.0%, 25.8%, and 10.3%, respectively. More patients undergoing urgent or emergency procedures (17.7% versus 7.8%; P =.03) subsequently developed pyrexia. However, there were no differences in wound infection (3.4% versus 8.3%; P =.13), positive cultures for respiratory (14.7% versus 11.4%; P =.16), urinary tract (5.2% versus 2.0%; P =.09), or other infection (8.6% versus 7.3%; P =.71) in patients experiencing postoperative pyrexia compared with those who did not. CONCLUSIONS: Pyrexia is common after cardiac surgery and resolves in the majority of patients by day 5. Because there is no association between early pyrexia and infection, diagnosis of early postoperative infection by pyrexia alone is insufficient and is better established by clinical assessment with microbiological evidence.

A method for descending thoracic aortic replacement retaining a posterior strip bearing intercostal vessels.

Operations for aneurysms of the descending thoracic aorta are still fraught with danger. Spinal cord injury remains a major cause of morbidity. Many therapeutic strategies have been suggested to reduce the incidence of this devastating complication, including reimplantation of intercostal vessels. However, reimplantation of intercostal vessels, both individually or in groups, is time consuming and compounded by the absence of a reliable means of identifying which vessels actually supply the cord. We present a technique that allowed inclusion of all potentially important descending aortic branching vessels into the repair leading to a favorable outcome in a series of patients.

A simple model to predict coronary disease in patients undergoing operation for mitral regurgitation.

BACKGROUND: Coexistent coronary disease can be identified in a third of patients with mitral valve disease. This study aims to evaluate candidate selection strategy using risk factor identification and logistic regression and to develop an additive model for the prediction of coexistent coronary disease. METHODS: The sample is a consecutive series of patients who had mitral repair from 1987 to 1999. Sensitivities and specificities were calculated for each risk factor. Variables for prediction of coronary disease were entered into a univariate analysis, and predictors were entered into a forward and backward stepwise multivariate logistic regression model to form a predictive score. An additive model was derived from transformation of the logistic model. Receiver operating characteristic curves were used to compare discrimination and precision quantified by the Hosmer-Lemeshow statistic. RESULTS: The American Heart Association and American College of Cardiology risk factor identification selection criteria for the 359 patients who had screening coronary angiography yielded 100% sensitivity and 1% specificity. Risk prediction with our logistic model produced a receiver operating characteristic curve area of 0.91 and Hosmer-Lemeshow score of 3.4 (p = 0.9). Similar discriminating ability for our patients was achieved by the Cleveland Clinic logistic model (receiver operator characteristic curve area of 0.79; Hosmer-Lemeshow score of 12; p = 0.1). Our five-item additive model produced receiver operating characteristic curve area of 0.91 and Hosmer-Lemeshow score of 3.81 (p = 0.80). CONCLUSIONS: Simple risk factor identification has excellent sensitivity but is limited by specificity. Logistic regression modeling is an accurate risk prediction method but is difficult to apply at the bedside. Simplicity and accuracy may be achieved by the logistic regression-derived simple additive model.

A versatile technique for aortic root replacement without pre-manufactured composite graft: a 12-year experience.

BACKGROUND AND AIM OF THE STUDY: Replacement of the aortic root is the treatment of choice for aneurysmal dilatation. Many modifications of the Bentall technique have been described, as have valve-sparing procedures. The study aim was to determine the outcome of a versatile modification of composite replacement that has been adopted over the past 12 years. Separate graft and prosthetic valve components were used to allow freedom of valve choice and the use of an appropriately sized graft for the distal aortic anastomosis. METHODS: Between January 1990 and March 2002, 59 patients (45 males, 14 females; mean age 56 +/- 14 years) underwent aortic root replacement using this technique. Indications for surgery were elective in 35 patients and emergent (usually type A aortic dissection) in 24. The range of valve prostheses used, their size, and the size of aortic graft used in each case was assessed. Durations of ischemia and cardiopulmonary bypass were recorded, as was postoperative blood loss and subsequent patient progress, including valve-related events, perioperative mortality and actuarial survival. RESULTS: A wide range of aortic graft sizes was combined with both mechanical and tissue valves (from 1-7 mm larger in diameter). Median postoperative blood loss was 550 ml (IQR 400-800 ml). Perioperative mortality was 5.1%. There were no valve- or technique-related deaths, and the median actuarial survival was 13.17 years. During a 12-year follow up there were no proximal aortic reoperations. CONCLUSIONS: This technique had favorable perioperative mortality, produced a secure proximal suture line, and allowed the surgeon free choice of both valve type and size of aortic graft. This minimized tension at the distal suture line, and produced good hemostasis, especially in those patients with fragile dissected tissues.

Porcine or human stentless valves for aortic valve replacement? Results of a 10-year comparative study.

BACKGROUND AND AIM OF THE STUDY: Stentless porcine valves in the aortic position exhibit similar excellent hemodynamic performance to homografts, but have the advantage of availability. Their performance was compared over a 10-year period in a single-surgeon and single-institution series. METHODS: Demographic, operative and mortality data were obtained retrospectively. Survivors were interviewed by telephone according to a defined protocol. Definitions and analyses were in accordance with joint STS/AATS guidelines. RESULTS: A total of 408 stentless porcine and homograft aortic valve replacements (AVR) was performed between 1991 and 2001. Five patients were excluded due to incomplete data, in addition to 82 patients who underwent AVR with a free-standing root replacement technique. Hence, 321 patients (217 males, 104 females; mean age 67 +/- 12 years) had a subcoronary implant. The median time to follow up was 4.9 years (range: 2.9-6.6 years). No differences were noted between homograft and stentless porcine valves in one- and five-year freedom from structural valve deterioration (99.1 versus 97.2% and 95.7 versus 93.1%; p = 0.10), reoperation (99.2 versus 99.4% and 97.8 versus 96.7%; p = 0.45) and endocarditis (98.3 versus 99.4% and 97.4 versus 99.4%; p = 0.14). Overall one- and five-year survival comparing homograft to stentless porcine valve was 90.4 versus 92.3% and 80.8 versus 73.7%, respectively; p = 0.23. Independent predictors of mortality on multivariate analysis were: ventricular function (p < 0.0001), increasing age (p < 0.001), increasing serum creatinine (p < 0.001) and concomitant coronary surgery (p = 0.05). Treated hypercholesterolemia was independently protective against mortality, with an odds ratio of 0.26 (CI 0.10 to 0.66; p = 0.005). CONCLUSION: The porcine stentless valve, when implanted in the subcoronary position, is an excellent alternative to the homograft and shows excellent clinical performance and durability at mid term.

The surgical treatment of bronchial carcinoma.

This article considers the current place of surgery in the treatment of bronchial carcinoma. Aspects of the diagnosis of this condition will be covered, but the main focus falls on the surgical procedures, their complications and the outlook for these patients according to tumour stage.